Methods and compositions for sequencing modified nucleic acids

Abstract

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

1 - 51 . (canceled) 
     
     
         52 . A method of generating a haplotype, the method comprising:
 a) providing a genomic DNA sample comprising a DNA fragment of at least 2 kb in length, wherein the DNA fragment comprises a region of interest;   b) subjecting the DNA fragment to a single-molecule sequencing reaction to generate a single sequence read that extends the full length of the DNA fragment comprising the region of interest, wherein the single sequence read comprises sequence information and kinetic information, wherein the kinetic information is indicative of modified bases within the DNA fragment;   c) analyzing the sequence information in the single sequence read to determine a base sequence for the region of interest;   d) analyzing the kinetic information in the single sequence read to identify modified bases within the region of interest; and   e) phasing the base sequence and the modified bases in the region of interest, thereby generating a haplotype that comprises both the base sequence of the region of interest and modified bases within the region of interest.   
     
     
         53 . The method of  claim 52 , wherein a plurality of fragments comprising the region of interest are each subjected to a single-molecule sequencing reaction within a single reaction mixture to generate a plurality of single sequence reads, and the analyzing further comprises generating a plurality of haplotypes for the region of interest. 
     
     
         54 . The method of  claim 53 , further comprising constructing a consensus haplotype sequence from the plurality of haplotypes. 
     
     
         55 . The method of  claim 52 , wherein the genomic DNA sample comprises one or more genomic DNAs selected from the group consisting of a human genomic DNA, a bacterial genomic DNA, a viral genomic DNA, a fungal genomic DNA, a forensic genomic DNA, a patient's genomic DNA, a diagnostic genomic DNA, a prognostic genomic DNA, a stem cell genomic DNA, a pluripotent cell genomic DNA, a parasitic organism genomic DNA, an embryonic genomic DNA, and a cancer cell genomic DNA. 
     
     
         56 . The method of  claim 52 , wherein the genomic DNA sample comprises both a maternal DNA fragment and a fetal DNA fragment from a single maternal blood sample, and further wherein a first haplotype is generated for the region of interest in the maternal DNA fragment and a second haplotype is generated for the region of interest in the fetal DNA fragment. 
     
     
         57 . The method of  claim 52 , wherein the genomic DNA sample comprises both a non-tumor-derived DNA fragment and a tumor-derived DNA fragment from a single patient sample, and wherein a first haplotype is generated for the region of interest in the non-tumor-derived DNA fragment and a second haplotype is generated for the region of interest in the tumor-derived DNA fragment, and further wherein differenced between the first haplotype and the second haplotype correspond to mutations that occurred during development of a tumor. 
     
     
         58 . The method of  claim 52 , wherein the genomic DNA sample comprises a pair of homologous chromosomes, the pair comprising a first homolog and a second homolog, and further wherein a first haplotype is generated for the region of interest in the first homolog and a second haplotype is generated for the region of interest in the second homolog. 
     
     
         59 . The method of  claim 52 , further comprising isolating the genomic DNA sample from a biological source selected from the group consisting of a blood sample, a sputum sample, a urine sample, a nasopharangeal sample, a vaginal sample, a biopsy, a forensic sample, a buccal sample, a patient sample, diagnostic sample, prognostic sample, embryonic sample, cancer genomic sample, a metagenomic sample, and a colonic sample. 
     
     
         60 . The method of  claim 59 , wherein the biological source is a metagenomic sample comprising a plurality of different types of microorganisms. 
     
     
         61 . The method of  claim 60 , wherein the microorganisms comprise at least one microbe selected from the group consisting of bacteria, archaea, fungi, viruses, and protozoa. 
     
     
         62 . The method of  claim 60 , wherein at least one of the microorganisms is a pathogenic organism. 
     
     
         63 . The method of  claim 52 , wherein the genomic DNA sample is not amplified, bisulfite-converted, or cloned prior to said single-molecule sequencing reaction. 
     
     
         64 . The method of  claim 52 , wherein the single-molecule sequencing reaction is selected from sequencing-by-incorporation, tSMS sequencing, and nanopore sequencing. 
     
     
         65 . The method of  claim 52 , wherein the single-molecule sequencing reaction has average read lengths in the 1000 to 10,000 base pair range. 
     
     
         66 . The method of  claim 52 , wherein the kinetic information in the single sequence read is indicative of patterns of modified bases within the DNA fragment. 
     
     
         67 . The method of  claim 52 , wherein the modified bases in the region of interest comprise at least one of 6-mA, 4-mC, 5-mC, hmC, phosphorothioate, and glucosylated hmC. 
     
     
         68 . The method of  claim 52 , wherein the region of interest is selected from the group consisting of a highly repetitive region, a euchromatic region, a heterochromatic region, an imprinted region, a pseudogene, a gene, and a regulatory region. 
     
     
         69 . The method of  claim 52 , wherein the read length of the single sequence read is at least 3000 base pairs in length. 
     
     
         70 . The method of  claim 52 , further comprising converting the genomic DNA sample into a sequencing library prior to said single-molecule sequencing reaction. 
     
     
         71 . The method of  claim 52 , wherein the genomic DNA sample comprises a gene and a pseudogene, and wherein a first haplotype is generated for the region of interest in the gene and a second haplotype is generated for the region of interest in the pseudogene, and further wherein the first haplotype and second haplotype comprise different patterns of modified bases.